TopoisomeraseIIDrugScreeningKitProductDescription

TopoIIinhibitorshavebeenshowntopossesseffectiveantibacterial,antiviralandanticanceractivity.IdentificationoftopoIIinhibitorsandantagoNISTsrequirestractablebiochemicalassaysemployingpurifiedenzyme. TheTopoisomeraseIIDrugScreeningKitcontainsthereagentsnecessarytoscreenforagentsthataffectenzymaticactivity. Toallowthecustomermaximumflexibility,purifiedtopoisomeraseIImaybepurchasedseparatelyfromthekit(TG2000H). Thiskitwillallowdetectionoftwoclassesoftopoisomeraseinhibitors:thosethatstimulateformationofcleavablecomplexesandthosethatantagonizetopoIIactionontheDNA. Alsoincludedinthiskitisdetaileddocumentationondesignandinterpretationofinhibitiondata. Knowninhibitorsareincludedascontrols. TheDNAsubstrate(pHOT1)includedinthisassayisidealforthesestudiesbecauseitcontainsasingle,highaffinitytopoIIcleavagesite(notethattopoIIisnotparticularlysequencespecific).TheassaysystemisbaseduponevaluatingtheformationofDNAcleavageproductswhichmaybenicked,opencircularDNAorlinearDNA.Theproductsarethenresolvedbyethidiumbromidegelelectrophoresisasdescribedinthekit.

TopoisomeraseIIDrugScreeningKitContents(representativeof100assaykitsize):

-SupercoiledDNA[pHOT1DNA]25ug
-MarkersarelinearpHOT1DNAandopencircularDNA
-10XTopoisomeraseIIassaybuffercomponents*

*ToimprovestABIlity,the10xTopoIIAssaybufferisprovidedastwocomponents: BufferA(noATP)andBufferB(ATP).TheComplete10xAssaybufferismadefresheachtimebycombiningBufferAandB.

-Sodiumdodecylsulfate(10%)
-10XGelloADIngbuffer(bromophenolblue,glycerol)
-Controlinhibitor,Etoposide
-ProteinaseK
-Detailedinstructionmanual

Note:TopoIIenzymeissoldseparately.

TopoIIDrugScreeningBackground:

ThisisacleavagebaseddetectionkitthatwillrevealwhetheraparticularagenteitherantagonizestheactionoftopoIIoraffectsthecleavage/religationcycle. Reactionsareassembledinmicrocentrifugetubesusingreagentssuppliedinthekit(buffer,DNA,controlinhibitor). ThiskitisdesignedtoworkwithpurifiedtopoisomeraseIIprovidedbyTopoGEN. Afteraddingenzyme,thereactionsareincubated15-30min(37°C),andterminatedbyadditionofSDS.

Atypicalreaction:

• H2O:Volumevariable,makeuptovolume(20ulinthiscase)
• 10XAssaybuffer2µl(madefreshfromBuffersAandB,seeKitdetails)
• DNA(supercoiled)1µl
• TestCompoundorcontrolinhibitor:Variable(limitedbysolventeffectsasaddressedincontrols)
• **PurifiedTopoisomeraseII:4-6Units(enzymesoldseparately)
• Incubate15-30minutesat37°C
• Add2µlSDS;digestwithproteinaseK(50ug/ml)
• Add0.1volsampleloadingdye(supplied)
• Loadgel:1%agarosecontaining0.5ug/mlethidiumbromidealongwiththemarkerDNAsincludedinthekit.

ThereactionsindicatedaboveeachlaneinthefigurewerecarriedoutusingtheenzymeprovidedbyTopoGENand1ulofDNA(0.25ugofformIpHOT-1,asprovidedineachkit). Thereactionproductswereanalyzedona1%agarosegel;therunningbuffercontained0.5ug/mlethidiumbromide. Thegelwasthendestainedindistilledwaterpriortophotodocumentation. Formationofthecleavablecomplex(linearDNAspecies)isclearlyvisIBLe. Additionally,relaxationofsupercoiledDNAsubstrate(formIpRYGDNA)wasinhibitedinallreactionsthatcontainedVM26. ThekitcontainsmarkerDNAs,VM-26controlinhibitor,allbuffersandcompleteinstructionsonusingthereagentstoscreenforinhibitorsoftopoisomeraseII. ThemanualspecifiesconditionsforgelelectrophoresistoresolveallDNAforms;publicationqualitygelswillresult.

**CleavagereactionsrequiremoretopoIIthanrelaxationassayssincetheformerisastoichiometricreactionofenzyme. Forexample,theabovereactionscontained4unitsofenzyme(unitsdefinedbydecatenationactivity).

ReviewsandCitations:

• Sudan,S.,&Rupasinghe,H.V.(2014).Flavonoid-EnrichedAppleFractionAF4InducesCellCycleArrest,DNATopoisomeraseIIInhibition,andApoptosisinHumanLiverCancerHepG2Cells.Nutritionandcancer,ahead-of-print(2014):1-10. doi:10.1080/01635581.2014.951733
• ChengMH,YangYC,WongYH,ChenTR,LeeCY,YangCC,ChenSH,YangIN,YangYS,HuangHS,YangCY,HuangMS,ChiuHF:B1,anoveltopoisomeraseIIinhibitor,inducesapoptosisandcellcycleG1arrestinlungadenocarcinomaA549cells. Anti-CancerDrugs2012,23(2):191-199. doi:10.1097/CAD.0b013e32834cd277
   
• ShiW,MarcusSL,LowaryTL:CytotoxicityandtopoisomeraseI/IIinhibitionofglycosylated2-phenyl-indoles,2-phenyl-benzo[b]thiophenesand2-phenyl-benzy[b]furans. BioorganicandMedicinalChemistry2010. doi:10.1016/j.bmc.2010.10.054
• GongY,FirestoneGL,BjeldanesLF:3,3'-DiindolylmethaneisanoveltopoisomeraseIIacatalyticinhibitorthatinducesS-phaseretardationandmitoticdelayinhumanhepatomaHepG2cells. MolecularPharmacology2006,69(4):1320-1327.

美国TopoGEN公司成立于1991年，是一家诊断公司，由一批在拓扑异构酶领域工作和在基础、临床、应用及相关方面研究经验丰富的研究者创建，为以药物开发为基础的拓扑异构酶和机制研发人员提供创新产品和服务。药物筛选试剂盒可以分析拓扑异构酶活性药物，用于癌症化疗和抗菌方面。TopoGEN公司还能提供原核和真核化验拓扑异构酶 (I型，II，IV和旋转酶)检测试剂盒，体外体内诊断均可应用。TopoGEN公司还提供DNA载体、DNA裂解目标、拓扑异构酶抑制剂、拓扑异构酶抗体和相关试剂盒等完整产品线。